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Endocr (2007) 31:72–81 Phytosterol Pygeum africanum regulates prostate cancerin vitro and in vivo Nader S. Shenouda Æ Mary S. Sakla Æ Leslie G. Newton ÆCynthia Besch-Williford Æ Norman M. Greenberg ÆRuth S. MacDonald Æ Dennis B. Lubahn Published online: 5 May 2007
Humana Press Inc. 2007 benign prostatic hypertrophy (BPH). More recently in the Prostate cancer is an important public health USA, the phytotherapeutic preparations of Pygeum afri- problem. It is an excellent candidate disease for chemo- canum and Saw palmetto have been marketed for prostate prevention because prostate cancer is typically slow health including prostate cancer prevention and treatment.
growing and is usually diagnosed in elderly males. Pygeum The anti-cancer potential of Pygeum africanum africanum (Prunus africana or Rosaceae) is an African has been tested both in vitro (PC-3 and LNCaP cells) and prune (plum) tree found in tropical Africa. An extract from in vivo (TRAMP mouse model).
the bark of Pygeum africanum has been used in Europe as a In tissue culture, ethanolic extracts (30%) of prevention and treatment of prostate disorders including Pygeum africanum inhibited the growth of PC-3 andLNCaP cells; induced apoptosis and altered cell kinetics;down regulated ERa and PKC-a protein, and demonstratedgood binding ability to both mouse uterine estrogen N. S. Shenouda
M. S. Sakla
L. G. Newton
receptors and LNCaP human androgen receptors. TRAMP D. B. Lubahn (&) mice fed Pygeum africanum showed a significant reduction Department of Biochemistry, University of Missouri-Columbia,920 East Campus Drive, 110A ASRC, Columbia, MO 65211, (P = 0.034) in prostate cancer incidence (35%) compared to casein fed mice (62.5%).
Pygeum africanum, which is widely used in Europe and USA for treatment of BPH, has a significant C. Besch-WillifordDepartment of Veterinary Pathobiology, University of Missouri- role in regulation of prostate cancer both in vitro and Columbia, Columbia, MO, USA in vivo and therefore may be a useful supplement forpeople at high risk for developing prostate cancer.
D. B. LubahnDepartment of Child Health, University of Missouri-Columbia,Columbia, MO, USA African herb
LNCaP
PC-3
TRAMP mice D. B. LubahnDepartment of Animal Sciences, University of Missouri- Columbia, Columbia, MO, USA Prostate cancer is an important public health problem Fred Hutchinson Cancer Research Center, Seattle, WA, USA accounting for more than 232,000 new cases and 33,000deaths in the USA in 2006 []. Prostate cancer is an R. S. MacDonaldDepartment of Food Science and Human Nutrition, Iowa State excellent candidate disease for chemo-prevention because University, Ames, IA, USA it is typically slow growing and is usually diagnosed inelderly males, therefore even a modest delay in the neo- plastic development achieved through pharmacological or MU Center for Phytonutrient and Phytochemical Studies,Columbia, MO, USA nutritional intervention could result in a substantial Endocr (2007) 31:72–81 reduction in the incidence of mortality []. The incidence affect membrane structure and function of tumor and host of prostate cancer is lower in Asian populations and in tissues, signal transduction pathways that regulate tumor vegetarians compared to populations consuming a more growth and apoptosis ]. The role of B-Sitosterol in prevention and treatment of BPH has been studied both in Components of the Western diet, most often cited as tissue culture and in clinical trials –however there playing a role in increased prostate cancer risk are animal are few studies on the role of B-Sitosterol in prostate products The most consistent correlation for prostate cancer. Von Holtz et al. [reported that B-Sitosterol cancer prevention is consumption of fruits, vegetables, and inhibited the growth of LNCaP cells and induced apop- grains, which are potential sources of phytoestrogens tosis via activation of the sphingomyelin cycle. Also Phytoestrogens are found in many plants which are com- Awad et al. [] found B-Sitosterol inhibited the monly used in traditional medicine. Phytoestrogens may growth and dissemination of PC-3 cells both in vitro and be either agonists or antagonists of estrogen receptors.
Therefore, it has been proposed that phytoestrogens may The aim of this work was to explore the possible influence prostate cancer cell growth and therefore herbal mechanisms by which Pygeum africanum regulates pros- therapies have been developed which include these com- tate cancer both in vitro (LNCaP and PC-3 human prostate pounds []. In vivo and in vitro studies have shown that cancer cell lines) and in vivo (in TRAMP Prostate Cancer phytoestrogens may influence not only steroid hormone Mouse model). These cell lines were selected to provide an metabolism and biological activity, but also intracellular androgen sensitive (LNCaP) and an androgen insensitive enzymes, growth factor action, protein synthesis, malignant (PC-3) comparison. To our knowledge, this is the first cell proliferation, and angiogenesis, thus making them report that extensively describes the anti-prostate cancer strong candidates for anticarcinogenic agents [].
activity of Pygeum africanum both in vitro and in vivo.
Pygeum africanum (Prunus africana or Rosaceae) is an African prune (plum) tree found in tropical Africa []. Thebark has been used in Europe since 1969 as a prevention and treatment of prostate disorders including benign pros-tatic hypertrophy (BPH). Also in Asia, and Africa, phyto- In vitro experiments therapy is considered a first line treatment for prostatedisorders and has been utilized effectively for centuries. In Pygeum africanum extract inhibited the growth of both the USA, phytotherapeutic preparations containing Pygeum PC-3 and LNCaP cells (Fig. ). The IC50 (the concentra- africanum and Saw palmetto are increasingly being mar- tion of the compound that is required to inhibit cell growth keted for the health of the prostate including prostate by 50%) was approximately 2.5 ll/ml (for both cell lines).
cancer prevention and treatment Phytochemical B-Sitosterol inhibited the growth of LNCaP cells but had investigations aimed at isolating the active ingredients of no effect on the growth of PC-3 cells (Fig. ).
Pygeum africanum have identified many compounds Cell proliferation was measured using a thymidine including fatty acids, sterols (B-Sitosterol and Campester- incorporation assay in the two prostate cancer cell lines ol) and pentacyclic triterpenoids ]. Multicenter clinical over 3 days incubated with and without the 2.5 ll/ml of trials have been done to study the role of Pygeum africa- Pygeum africanum. In PC-3 and LNCaP cells, Pygeum num in prevention and treatment of BPH –however africanum inhibited growth by 50% from day 1 through one study on the mechanism of action of Pygeum africa- day 3 (Fig. ).
num in prostate cancer has been described [The major In both prostate cancer cell lines, a significant increase effects of Pygeum africanum have been reported to be in the number of cells in the S phase of the cell cycle was caused by its main active ingredient, B-Sitosterol which is induced by Pygeum africanum in PC-3 (Fig. and present in high concentrations in the Pygeum africanum LNCaP (Fig. D) cells when compared to untreated cells (Fig. A, C).
B-Sitosterol, a sterol related to cholesterol, is present in Pygeum africanum at 2.5 ll/ml induced significant three commonly used prostate herbal supplements, Pyge- apoptosis in both cell lines (PC-3 and LNCaP) when um africanum, Saw palmetto, and Hypoxis. These three compared with untreated cells (Table ).
herbs are commonly used in commercial supplements for Pygeum africanum and B-Sitosterol were tested for their prostate health [Epidemiological and experimental potential to displace bound estradiol in mouse uterine studies [] suggest that dietary phytosterols may offer cytosol using the competitive 3H-estradiol ligand-binding protection from the most common cancers in Western assay. At 5 ll/ml Pygeum africanum displaced >70% of societies, such as colon, breast and prostate cancer. It has 3H-estradiol; however, B-Sitosterol (5 lM) did not com- been proposed that phytosterols such as B-Sitosterol pete for 3H-estradiol binding (Fig. Endocr (2007) 31:72–81 Concentration (μl/ml) Concentration (µM) Concentration (μl/ml) Fig. 1 PC-3 (A) and LNCaP (B) cells were plated at 1 · 104 cells per Concentration (μM) well in 24 well dishes and grown to 50% confluence. After 2 days themedia was replaced with complete media containing 0–10 ll/ml of Fig. 2 PC-3 (A) and LNCaP (B) cells were plated at 1 · 104 cells per Pygeum africanum (30% ethanol extract) and incubated for 72 h. The well in 24 well dishes and grown to 50% confluence. After 2 days total cellular protein concentration was determined by DC Bio-Rad the media was replaced with complete media containing 0–25 lM B- assay. The IC50 for Pygeum africanum was calculated based on the sitosterol loaded on cyclodextrin (CD) and incubated for 72 h. The growth inhibition after 72 h in comparison to the zero treatment total cellular protein concentration was determined by DC Bio-Rad control. Values are means ± SEM of three individual experiments assay. The IC50 for B-sitosterol was calculated based on the growth (Each individual experiment was performed in duplicate) inhibition after 72 h in comparison to the zero treatment control.
Values are means ± SEM of three individual experiments (Eachindividual experiment was performed in duplicate) Pygeum africanum and B-Sitosterol were tested for their There was no significant effect of diet on total body potential to displace bound DHT in LNCaP cell cytosol weight, reproductive tract, testes or prostate weights using the competitive 3H-DHT ligand-binding assay. At (Table ). The large variation in the prostate weight in the 5 ll/ml Pygeum africanum displaced >60% of 3H-DHT; in vivo treatment group was because one of the tumors in however, B-Sitosterol (5 lM) was not able to compete for PDC stage of the Pygeum africanum fed group was huge.
3H-DHT binding (Fig. The difference in PDC incidence was still not statistically Pygeum africanum showed a dose dependent inhibition significant (P > 0.05).
of ERa protein expression in PC-3 cells (Fig. AlsoPygeum africanum at 2.5 ll/ml inhibited PKC-a expres-sion in LNCaP but not in PC-3 cells (Fig. In vivo experiments In the present study, an extract of Pygeum africanum in-duced 50% growth inhibition of both PC-3 and LNCaP Among TRAMP mice fed Pygeum africanum extract for cells. This was in agreement with Margalef et al. who 5 months, fewer animals developed prostate cancer (35%) reported growth inhibition of prostate cancer-derived cells than mice fed the casein diet (62.5%). The largest effect (LNCaP and CA-HPV-10). The inhibition of the growth was on WDC incidence (P = 0.034; Table There was a of PC-3 and LNCaP cells was present from day 1 through non-significant effect on PDC ‘‘neuro-endocrine like car- day 3 of incubation with the Pygeum africanum extract.
cinoma incidence'' (P = 0.78).
The effect of Pygeum on the prostate cancer cell lines was Endocr (2007) 31:72–81 signal transduction pathways that regulate tumor growth, and apoptosis However, we found no effect of B-Sitosterol on PC-3 cell growth but we did see an effect similar to Awad et al. ] who showed that B-sitosterol inhibited the growth of LNCaP cells in vitro. However, in contrast to our results Awad et al. found an effect of B-Sitosterol on PC-3 cell growth in 5% non-stripped serum ].

stn 10000
The present study showed that Pygeum africanum at 2.5 ll/ml induced an accumulation of the cells in the S phase of the cell cycle for both PC-3 and LNCaP cells, as well as significant apoptosis compared to untreated cells.
These results differed from Margalef et al. [who re- ported a block in the transition from G1 to S phase in the cell cycle of PZ-HPV7 cells with an ethanolic extract of Pygeum africanum.
Interest in the physiological roles of bioactive com- pounds present in plants has increased dramatically over the last decade. Of particular interest because of their re- ported roles in human health is the class of compounds known as phytoestrogens. Phytoestrogens embody several groups of non-steroidal estrogens that are widely distrib-uted within the plant kingdom [The phytoestrogen genistein for example binds to ER with a preference for the more recently described ER b ]. In the present study,Pygeum africanum extract displaced 3H-estradiol in a Fig. 3 PC-3 (A) and LNCaP (B) cells were plated at 1 · 104 cells per competitive ligand-binding assay using mouse uterine well in 24 well dishes and grown to 50% confluence. After 2 days themedia was replaced with complete media containing 2.5 ll/ml of cytosol. This was in agreement with Mathe [, who Pygeum africanum (30% ethanol extract) and incubated for 72 h. The reported that Pygeum africanum had a phytoestrogenic total DNA concentration was determined at day 1, 2 and 3 by effect. Also, Pygeum africanum at the same concentration thymidine incorporation assay. Values are means ± SEM of three displaced 3H-DHT in a competitive ligand-binding assay individual experiments (Each individual experiment was performed induplicate) using LNCaP cell cytosol. The androgen receptor bindingaffinity of Pygeum africanum might help to explain the not a toxic effect as we saw no effect of Pygeum africanum inhibitory effect on 5 alpha reductase reported by Rhodes on the CaCO2 (Colon Cancer) cell line that has similar et al. [Recently some phytoestrogens (genistein and metabolic activity and cell cycle time, at concentrations up daidzein) have been found to induce apoptosis and inhi- to 20 ll/ml of the ethanolic extract of Pygeum africanum bition of the PC-3 and LNCaP cell growth through their (Data not shown).
binding and regulation of both ER and AR. Cao et al ] Phytochemical investigations aimed at isolating the ac- found that the expression of the ER-alpha and ER-beta tive ingredients of Pygeum africanum have been exten- genes decreased, while AR gene was not expressed, after sively undertaken. Many compounds have been identified incubation with genistein and daidzein in PC-3 cells. Also, including fatty acids, sterols (B-Sitosterol and Campester- they reported that the apoptosis percentage of LNCaP cells ol) and pentcyclic triterpenoids ]. B-Sitosterol, as one of was elevated significantly by daidzein.
the marker compounds for Pygeum africanum, has been The effects of estrogens on prostate cancer have been tested for its ability to inhibit the growth of both PC-3 and reviewed by Ho [and by Steiner and Raghow [ LNCaP cells , Few studies on the role of B-Sitos- Estrogen therapy, principally the use of diethylstilbestrol terol in prostate cancer have been described. We found that (DES), is a suggested practice in prostate tumor treatment.
B-Sitosterol inhibited the growth of LNCaP cells. This Its primary mode of action is through feedback on the agrees with Von Holtz et al. [who reported that B- anterior pituitary with suppression of gonadotropin secre- Sitosterol inhibited the growth of LNCaP cells and induced tion and subsequent decrease in testosterone production by apoptosis via activation of the sphingomyelin cycle or by Leydig cells of the testis. This in turn causes a decrease in regulation of the activity of PKC-alpha [Also Awad androgens required for hormone dependent cancer. How- et al. ] reported that B-Sitosterol might affect mem- ever, direct effects of DES in the prostate are also possible.
brane structure and function of tumor and host tissues, Whether DES works directly in the prostate through a

Endocr (2007) 31:72–81 Fig. 4 Cell cycle changes inuntreated PC-3 and LNCaP cells(A & C) and PC-3 and LNCaPcells treated with 2.5 ll/ml ofPygeum africanum (B & D)were detected by flowcytometry. The untreated PC-3and LNCaP cells showednormal cell cycle events (A &C) while the cells treated with2.5 ll/ml of Pygeum africanumshowed accumulation of cells inS phase of the cell cycle(B & D). M1 ¼ G1 phase; M2 ¼ S phase; M3 ¼ G2/M Table 1 Induction of apoptosis by the Pygeum africanum extract in PC-3 and LNCaP cell lines as determined by the TUNEL assay LNCaP (% apoptotic PC-3 (% apoptotic Mean ± SEM. Values are percent of total events counted. Values within each column with different letter superscripts are significantlydifferent, P < 0.05 or less. N = 3 individual experiments and each experiment was performed in duplicate. Negative control is theuntreated cells. Positive control is camphotechin treated cells No Hormone
Pygeum 5 µl/ml
B-sitosterol 5 µM
Fig. 5 ER Saturation Binding Analysis—WT uterine cytosol was classical ERa pathway, through ERb or another estrogen- incubated overnight at 4C with 3H-estradiol (5 nM final concentra- dependent mechanism has not been fully determined. The tion) and the aliquot of Pygeum africanum (5ll/ml of 30% ethanol present study showed a dose dependent inhibition of ERa extract) and B-sitosterol (5 lM). Bound and free ligands wereseparated by dextran-coated charcoal and an aliquot of bound protein expression in PC-3 cells which express a significant radioactivity was measured by scintillation counting. Values are amount of ERa protein This was in agreement with means ± SEM of three individual experiments (Each individual Mathe [, who reported that Pygeum africanum had experiment was performed in duplicate). Bars designated with a phytoestrogenic effect on the prostate resulting in a sig- asterisks are significantly different from no hormone, P < 0 05 or less nificant effect on the morphology of the glandular epithe-lium of the mouse prostate presumably due to the effect of The present study showed that TRAMP mice fed Pyg- eum africanum in a dose of 0.128 gm/kg of the diet showed A recent review identified protein kinase C-a (PKC- a), a a significant and marked reduction of WDC tumor inci- serine–threonine kinase involved in cellular signaling path- dence (35%) compared to mice fed casein diet (62.5%).
ways, as a target to modulate prostate cancer. PKC-a has This could be explained by Pygeum africanum potentially been found in both hormone insensitive (PC-3) and hormone regulating prostate cancer in vivo through several mecha- sensitive (LNCaP) prostate cancer cells [The present nisms including ERa, AR, PKC-a and apoptosis-related study found that Pygeum africanum at 2.5 ll/ml inhibited the mechanisms. The mice fed Pygeum africanum did not expression of PKC-a in LNCaP but not in PC-3 cells.
show any significant difference from the mice fed casein in

Endocr (2007) 31:72–81 terms of their total body weight, reproductive tract, testesand prostate weights. This finding confirms the absence of any obvious toxic effects among the mice fed Pygeum africanum at this dose.
In conclusion, this study showed that Pygeum africanum which is widely used in Europe and the USA for treatment M rep 1500
of BPH might be able to play a significant role in regulation of prostate cancer both in vitro and in vivo. Thus, Pygeum africanum may be of dual benefit to men who are at highrisk of developing either BPH or prostate cancer.
No Hormone
DHT 100nM
Materials and methods Fig. 6 AR Saturation Binding Analysis—LNCaP cell cytosol wasincubated overnight at 4C with 3H-DHT (5 nM final concentration) Pygeum africanum bark was obtained from Nutraceutical and the aliquot of Pygeum africanum (5ll/ml of 30% ethanol extract) Corp./Solaray (Park City, UT) and was by weight 13% and B-sitosterol (5 lM). Bound and free ligands were separated bydextran-coated charcoal and an aliquot of bound radioactivity was total sterols. Ethanolic extracts (30% W/V) were made for measured by scintillation counting. Values are means ± SEM of three tissue culture experiments and stocks were kept at –20C.
individual experiments (Each individual experiment was performed in Human reactive polyclonal antibody for ERa (PA1–308) duplicate). Bars designated with asterisks are significantly different and PKC-a (PA1–405) were purchased from Affinity Bio from no hormone, P < 0 05 or less Reagents (Golden, CO). B-Sitosterol (98% pure) was ob-tained from Sigma Chemical Co. (St. Louis, MO) and usedin tissue culture after being loaded on Cyclodextrin (CD)vehicle to be accessible by the cultured cells [Unla-beled 17b estradiol and DHT were obtained from SigmaChemical Co. (St. Louis, MO). 3H-17b estradiol and 3H-DHT were obtained from Amersham Pharmacia Biotech(Piscataway, NJ).
Fig. 7 Western blot analysis of ERa protein expression in PC-3 cellstreated with different concentrations of Pygeum africanum using human reactive polyclonal antibody. Extracts of PC-3 cell line were resolved by gel electrophoresis, electro-blotted onto nitrocellulosemembrane, and immuno-detected as described in Materials and Cell culture experiments Methods. Lane 1, 2 and 3 extracts of PC-3 cells treated with Pygeumafricanum (zero treatment control, 1 and 2.5 ll/ml of 30% ethanolextract); lane 4, protein standard; lane 5, MCF-7 cells as a positive Human prostate carcinoma (PC3 and LNCaP) cells were obtained from the ATCC and cultured in complete RPMI1640 medium (Gibco Rockville, MD) supplemented with10% fetal bovine serum (FBS), penicillin (100 units/ml)streptomycin (100 lg/ml)), L-glutamine (5 mM)), NEAA(0.1 mM Non Essential Amino Acids), HEPES buffer(10 mM), glucose (2.5 gm/l) in 5% CO2 at 37C.
Growth inhibition curve and IC50: PC3 and LNCaP cells were plated at 1 · 104 cells per well in 24 well dishes withRPMI 1640 complete culture medium and cells were grownto 50% confluence. After 2 days the media were replacedwith complete media with charcoal stripped serum con- Fig. 8 Western blot analysis of PKC-a protein expression in PC-3 taining 0–10 ll/ml of the Pygeum africanum extract or and LNCaP cells treated with different concentrations of Pygeum 0–25 lM of B-Sitosterol and incubated for 72 h. The media africanum using human reactive polyclonal antibody. Extracts of PC- were aspirated, cells washed twice with phosphate buffered 3 and LNCaP cell lines were resolved by gel electrophoresis, electro-blotted onto nitrocellulose membrane, and immuno-detected as saline (PBS) (10 mM, pH 7.4), and 1N NaOH (250 ll) described in Materials and Methods. (A) Lane 1, 2 and 3 extracts added to lyse the cells. The total cellular protein concentra- of PC-3 cells treated with Pygeum africanum (zero treatment control, tion was determined by DC Bio-Rad assay using the manu- 1 and 2.5 ll/ml of 30% ethanol extract). (B) Lane 1, 2, and 3 extracts facturer's protocol (Bio-Rad laboratories, Hercules, CA).
of LNCaP cells treated with Pygeum africanum (zero treatmentcontrol, 1 and 2.5 ll/ml of 30% ethanol extract) The IC50 is the concentration where 50% of the maximum Endocr (2007) 31:72–81 Table 2 Effect of Pygeum africanum on the incidence of prostate tumorigenesis in ERaWT/TRAMP mice Pygeum africanuma There was a statistically significant difference (P = 0.034) between the Pygeum africanum and casein diet regarding prostate tumorigenesis Table 3 Body and organ weights of mice from different dietary groups a Means ± SEM. No significant differences were seen between treatment groups inhibition is observed. To calculate IC50, we first take the Detection of apoptosis using TUNEL assay: PC3 and difference between the protein value of the maximum inhi- LNCaP cells were plated at 1 · 106 cells in 100-mm cul- bition (where the growth curve levels out) and the protein ture dishes in RPMI 1640 complete media and grown to value of no inhibition at zero concentration. This difference 50% confluence. After 2 days the media was replaced with is multiplied by 0.5 and then the resulting protein value is complete media containing 2.5 ll/ml of Pygeum africanum subtracted from the no inhibition protein value to obtain the extract and incubated for 72 h. The adherent cells were protein value that corresponds to the IC50 concentration of released with trypsin, washed with PBS and processed for the extract being used. 100% ethanol was used in the treat- labeling with fluorescein-tagged deoxyuridine triphosphate ment control group up to 1% final concentration without any nucleotide and propidium iodide by the use of an APO- observed cell toxicity.
Direct Apoptosis Kit obtained from Phoenix Flow Systems Time course study using thymidine incorporation assay: (San Diego, CA). The labeled cells were then analyzed by PC3 and LNCaP cells were plated at 1 · 104 cells per well flow cytometry.
in 24 well dishes with RPMI 1640 complete culture med-ium and cells grown to 50% confluence. After 2 days the media was replaced with complete media containing2.5 ll/ml of Pygeum africanum extract and incubated for 3H-estradiol binding assay: Tissue cytosol preparation 24, 48 or 72 h. The cellular proliferation was determined (WT mouse uterine cytosol) and estradiol competition by thymidine incorporation assay [ binding analysis of Pygeum africanum extract were per- Cell cycle kinetics: PC3 and LNCaP cells were plated formed as previously described [].
at 1 · 104 cells per well in 24 well dishes with RPMI 1640 3H-DHT binding assay: The capacity of pygeum afri- complete culture medium and cells grown to 50% canum extract to bind androgen receptor was assessed by confluence. After 2 days the media was replaced with DHT competition binding assay using LNCaP cell cytosol complete media containing 2.5 ll/ml of Pygeum africanum as previously described [ extract and incubated for 72 h. The adherent cells werereleased with trypsin, washed twice with phosphate buf-fered saline (PBS) and collected by centrifugation. The cell Western blotting for ER detection pellet was resuspended in 2.5 ml of cold ethanol (95%) for1 h at 4C, washed with PBS, and incubated with 5 ll Expression levels of both ERa and PKC-a in PC-3 and RNase (20 lg/ml final concentration) at 37C for 30 min.
LNCaP cells treated with IC50 of Pygeum africanum were The cells were chilled on ice for 10 min then stained with quantified by western immunoblot as previously described propidium iodide (50 lg/ml final concentration) for 1 h We have controlled for the decrease in cell number and analyzed by flow cytometry (CyAn Flow, Cytomation, through cell cycle inhibition and apoptosis by loading the Inc, Somerville, NJ).
same amount of protein in each lane (20 lg).

Endocr (2007) 31:72–81 In vivo experiments was used, with ambient temperature and humidity set at21C, and 50%, respectively. Animals were monitored Prostate cancer model weekly for body weight and tumor burden and wereeuthanized after 5 months.
Animal protocols followed were approved by the Univer-sity of Missouri Animal Care and Use Committee and Dietary Pygeum africanum treatments followed NRC guidelines. All breeder pairs were main-tained on a casein-based diet (AIN 93G) as previously All breeder pairs were maintained on the casein-based diet described ]. This model, known as TRAMP ] for through pregnancy and lactation. At 6 weeks of age, male TRansgenic Adenocarcinoma of the Mouse Prostate, was TRAMP mice were randomly assigned to either the casein- developed by placing the SV40 early genes encoding T and based diet (AIN 93G) or to the same diet to which Pygeum t antigens under the control of the rat probasin promoter, africanum was added. The concentration of Pygeum afri- which has been shown to be highly and specifically ex- canum was chosen in reference to the human consumption pressed in the mouse prostate [The probasin (PB) dose in BPH [, The mouse diet formulation contained promoter is androgen and zinc regulated, with two andro- 0.128 grams Pygeum africanum/kg diet, which was higher gen response elements located in this region. PB is local- than the recommended human dose in BPH but well within ized in the ducts and nuclei of prostate epithelial cells, yet the range attainable through supplements. The bark of PB's function has not been identified. The transgene, Pygeum Africanum was grounded and added as a dry known as PBTag, is highly expressed in the dorsal and powder, the diet mixed in a Hobart mixer and pelleted ventral prostate lobes. This oncoprotein is known to abro- by dry, low heat extrusion into pellets. Animals were gate tumor suppressor functions of p53 and Retinoblastoma given continual access to the diet and body weights were (Rb) and thereby drive the cell through the normal cell recorded weekly.
cycle checkpoints. Mutations in p53 are important in the At termination the reproductive tract (testes, vas defer- prostate and occur in one third of early stage prostate ens, empty urinary bladder, seminal vesicles and prostate cancers [In our hands 60–85% of TRAMP mice lobes), testes and prostates were removed and weighed. At develop prostate cancer spontaneously within 5 months.
the time of collection (5 months), a portion of each prostate The C57BL6/J TRAMP mice were housed in pairs in was fixed in neutral buffered formalin for histological breeder boxes with micro-isolator lids and given free ac- analysis and the remainder snap-frozen in liquid nitrogen cess to food and water. A daily light: dark cycle of 12:12 h and stored at –80C.
Fig. 9 Stages of cancer in the TRAMP mouse prostate (H&E stained thickened muscle wall. (D) Well-differentiated adenocarcinoma with sections, bar = 50 mm). (A) Normal prostate tubule (dorsal prostate) increased cribriform lesions, invasion of tubule wall and increased with single epithelial layer and thin muscle wall. (B) Hyperplastic mitotic rate in epithelium. (E) Moderately well-differentiated prostate tubule with more numerous epithelial cells but retention of adenocarcinoma with stratification or layering of epithelium and loss simple layer and thin muscle wall. (C) Prostate tubule with prostate of formation of tubules. (F) Poorly differentiated carcinoma with intraepithelial neoplasia (PIN) and cribriform hyperplastic lesions and Endocr (2007) 31:72–81 In vivo experiments All lobes of the prostate glands were examined for nodular Prostate histology was classified as non-cancer stages expansion, excised, and weighed. If their appearance was (Normal, Hyperplasia and PIN) or cancer stages (WDC, normal, then one dorsal lobe and all other lobes of the MDC, and PDC or neuro-endocrine like carcinoma). Tu- prostate were snap-frozen in liquid nitrogen and subse- mor incidence data was analyzed using X2 test. Values that quently stored at –80C. If nodules or abnormal findings achieved P < 0.05 were considered to be significantly were observed in regions of the prostate other than the different. Comparison within the same tumor stage be- dorsal lobe, the lesions were bisected, with half of the tween the two dietary groups has also been done. Body sample fixed for histological evaluation and the other half weight, reproductive tract weight, testicular weight, and snap-frozen. The other dorsal prostate lobe and any prostate weight were analyzed using a two-sample t-test, abnormal prostate lesions were immersed in 10% neutral assuming unequal variance.
buffered formalin for 12–18 h, transferred to 70% ethanoland then processed for paraffin embedment. Paraffin- We would like to thank Dr. Wendy Applequest, Missouri Botanical Gardens for her kind assistance in obtaining the embedded tissues were cut in 4 micron sections and stained Pygeum africanum extract. We appreciate the help of Dr. George with hematoxylin and eosin.
Rottinghaus, Department of Analytical chemistry, University of One or more sections of the dorsal prostate lobe were Missouri-Columbia in making the ethanol extract of Pygeum africa- assigned random numbers so as to blind the pathologist.
num. This work was supported by the Missouri University Center forPhytonutrient and Phytochemical Studies (MUCCPS), NIH Grant # Prostate sections were examined and graded according to P01-ES510535 and NIH grant # R01AT002978.
morphologic criteria established for mouse models ofprostate cancer []. The proliferative changes of thesecretory epithelia of the prostate tubules were recorded,scored by independent veterinary pathologist who were unaware of the treatment groups and assigned to one of thefollowing designations: (A) normal, (B) hyperplastic, (C) 1. A. Jemal, R. Siegel, E. Ward, T. Murray, J. Xu, C. Smigal, M.J.
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